The long-term goal of this project is to understand the processes involved in the evolution of antibiotic resistance genes and their dissemination to populations of pathogenic bacteria. There is evidence that at least some antibiotic resistance genes have originated in streptomycetes--developmentally, morphologically, and biochemically complex antibiotic-producing bacteria that are chromosomally resistant to the antimicrobials they synthesize, and which can transfer segments of chromosomal DNA to both circular and linear plasmids. The proposed research, which continues and extends an ongoing multifaceted effort, is aimed at: 1) elucidating the role of pleiotropic and pathway-specific regulators of morphological differentiation, antibiotic biosynthesis, and antibiotic resistance in S. coelicolor and S. lividans on a genome- wide basis, 2) identifying, characterizing, and elucidating the mechanism of action of, and cellular targets of, small regulatory RNAs (sRNAs) of Streptomyces, and 3) elucidating mechanisms involved in the evolution and propagation of linear plasmids. The research will employ a combination of genetic and biochemical approaches, including mutational analysis of--and overexpression of--genes in regulatory pathways, DNA microarray studies of expression of Streptomyces genes and small intergenic RNAs, protein purification and analysis, and genetic analysis of linear extrachromosomal replicons. Collectively, the proposed studies will provide information that potentially will be useful in limiting the natural spread of antibiotic resistance genes, in developing antimicrobials that circumvent existing resistance mechanisms, and in combating antibiotic resistance that may be introduced intentionally, as acts of bioterrorism, into populations of pathogenic or toxigenic bacteria.